Squaraine dyes are organic dyes with very intense fluorescence spectra, typically in the red and near infrared region, which makes them attractive for biological applications. They are characterized by an aromatic four-membered ring system derived from squaric acid.
Some squaraine dyes may be conjugated to biomolecules through a free carboxylic acid group. However, these lipophilic squaraine dyes suffer from three problems in physiological solutions: poor solubility, massive aggregation-induced loss of fluorescence signal, and poor stability. Even sulfonated squaraine derivatives that are water-soluble are still unstable in PBS.
Most squaraines are encumbered by nucleophilic attack of the central four-membered ring, which is highly electron deficient. This encumbrance may be attenuated by trapping the squaraine inside a macrocycle to form a permanently interlocked molecule called a squaraine rotaxane. Formation of a rotaxane around the dye helps to protect it from nucleophiles, which improves the stability of squaraine dyes in physiological solutions like phosphate buffered saline (PBS). However, squaraine rotaxanes still lose fluorescence signal under very strong aggregation conditions such as phosphate buffered saline, which limits their usefulness in biological applications.